The present invention relates to a method for treating an aqueous flow colonised by Legionella by applying a pulsed electric field to the flow, to a method for treating an aqueous flow by electropulsing, and to its application to eliminating Legionella
Legionella are Gram negative bacilli that cause a potentially serious pneumonopathy known as xe2x80x9clegionnaire""s diseasexe2x80x9d and a benign flu-like syndrome known as xe2x80x9cPontiac feverxe2x80x9d. It is estimated that the annual number of cases of legionnaire""s disease in France is about 3000, of which 400 to 500 have been confirmed by the Centre Nationale de Rxc3xa9fxc3xa9rence [National Reference Centre].
These bacteria multiply in aqueous media, more readily when the temperature is between 30xc2x0 C. and 40xc2x0 C., with survival becoming difficult beyond 50xc2x0 C. Contamination can occur by inhaling water micro-droplets containing such bacteria, in particular when using hot water for sanitary purposes, mainly from showering and via air conditioning units. In this case, it is not so much the air taken into an air conditioned building that can transport the bacterium (except in the case of dysfunction such as poor positioning of the external air intake), but rather the stream that leaves the coolant tower, generally located on the roof of the building.
With a mortality rate that can be as high as 10%, this disease poses a difficult problem as regards prevention, in particular in a hospital where infirm people can develop the disease more easily, and in thermal facilities, where the water cannot be treated using conventional means.
When a case of legonnaire""s disease is diagnosed, a search is made by the services responsible for the contaminating source. When a positive response is obtained, the unit must be decontaminated, which in the example of hot water systems for sanitary purposes, involves several steps in which the boiler temperature is raised (70xc2x0 C.) followed by xe2x80x9cflushingxe2x80x9d the plumbing after isolating the circuit in question. Chlorinating with high residual free chlorine levels can also be carried out, independently or combined with the first technique. Unfortunately, in the absence of xe2x80x9ccontinuousxe2x80x9d treatment, recolonisation of the system in question is observed over subsequent weeks in the majority of cases.
There is a need for a method and units that can destroy such bacteria effectively, without involving a deleterious secondary effect, such as a risk of bums or the toxic effects of chlorine and which can be operated permanently in an economic manner.
Applying an electric field to cells has already been described: when a cell is placed in an electric field, it distorts the field lines, causing an accumulation of charge on the cell surface. This results in an induced transmembrane potential difference xcex94V which is superimposed on the native difference xcex94xcexa80 [Bernhardt J. and Pauly H. (1973): (2)].
The most complete formula used in the case of a field with square wave kinetics and a spherical cell in suspension is as follows [Sale A. J. H. and Hamilton W. A. (1967): (18); Tsong T. Y. et al., (1976): (24); Kinosita K. and Tsong T. Y. (1977a) (9)]:
xcex94Vt=fg(xcex)rEt cos xcex8(1xe2x88x92exe2x88x92t/xcfx84p) 
The expression for this potential difference induced at a point M at time t is a function of:
Et: the intensity of the applied electric field at time t;
f: the form factor for the cell (1.5 in the case of a sphere);
g(xcex) the factor for membrane permeability xcex;
r: the cell radius;
xcex8: the angle between the macroscopic electric field vector and the normal to the plane of the membrane at the point considered, M;
xcfx84p: the charge time for the membrane capacity (of the order of one microsecond);
t: time of application of field.
When the pulse duration is much longer than the time to charge the membrane (t greater than  greater than xcfx84p), the term (1xe2x88x92exe2x88x92t/xcfx84p) tends towards 1 to give the stationary state of the conventional formula:
xcex94Vt=fg(xcex)rEt cos xcex8
The term in cos xcex8 indicates that for a given field, the amplitude of this potential difference is not identical at every point of the cell. It is a maximum at points facing the electrodes (poles) and reduces along the cell surface to become zero at the equator
This potential difference generated by the field is added to the native potential difference xcex94xcexa80. This produces a resultant potential difference xcex94Vr.
xcex94Vr=xcex94xcexa80+xcex94V 
For the cellular hemisphere facing the anode, the numerical values of xcex94xcexa80 and xcex94V add to take into account the vector of the field effect, causing membrane hyperpolarisation. In contrast, for the hemisphere facing the cathode, the numerical values of xcex94xcexa80 and xcex94V subtract and the membrane undergoes depolarisation.
When this resulting membrane potential difference exceeds a threshold value estimated to be 200-250 mV [Teissixc3xa9 and Tsong (1981): (20)], a permeabilisation phenomenon is induced [Neumann E. and Rosenheck K. (1972): (13); Kinosita K. and Tsong, T. Y.: (1977b) (10)].
The membrane structure responsible for this membrane permeability is unknown at the present time, and the term xe2x80x9ctransient permeabilisation structurexe2x80x9d (TSP) is preferentially used, which is usually expressed by the term xe2x80x9cporesxe2x80x9d.
Under particularly drastic electropulsing conditions, electropermeabilisationis an irreversible phenomenon that leads to cell death, or electromortality, in particular in the case of microorganisms [Hamilton and Sale (1967): (5); Sale and Hamilton (1967): (18): Hxc3xclsheger et al., (1981): (6), (1983): (7); Mizuno and Hori, (1988): (12); Kekez et al., (1996): (8); Grahl Mxc3xa4rkl, (1996): (4)]. This property has been used either to lyse cells to recover a metabolite of interests not naturally excreted by the cell, or to eradicate cells from the environment (disinfecting) or from alimentary fluids (non thermal sterlisation) [Knorr et al., (1994): (11); Qin et al., (1996): (15); Qin et al., (1998): (16)].
Electromortality can occur immediately after electropulsing (short term mortality), or over a longer time period.
Applying pulsed electric fields to cell cultures is known, in particular in a fixed bed [Sale and Hamilton (1967): (18)].
Under those conditions, the sensitivity of the cells is known to depend in particular on the nature and geometry of the electrodes and on the electrical conditions employed (field intensity, number, form and duration of pulses) and on the composition of the medium.
Two pulse systems exist, depending on the volume treated: a fixed bed pulse system, known as a batch system, which can only treat small volumes that depend on the dimensions of the electrodes, and a flow pulse system that can treat a flowing cell suspension. Regarding the flow method, two strategies have been described: continuous flow and sequential flow. In the second model, the pulse chamber is filled, the flow is stopped, the field is applied and the chamber is then emptied.
This sequential flow model was developed for electrofusion work where the contact is mediated by dielectrophoresis [Bates et al., (1983): (1) (Zachrisson and Bonman (1984): (25)].
The advantage of a flow system is that it can be used to treat large volumes.
Usually, flow systems are used wherein the field is perpendicular to the flow [Teissixc3xa9 et al., (1988) (22); Teissixc3xa9 and Rols, (1988): (21); Sixou and Teissixc3xa9 (1990): (19); Teissixc3xa9 et al., (1992): (23); Rols et al., (1992): (17); Bruggemann et al., (1995): (3); Qin et al., (1996): (15)]. Systems with coaxial electrodes produce a non uniform field that is also perpendicular to the flow [Qin et al., (1996): (15); Qin et al., (1998): (16)].
When treating flowing streams, the pulses can be in a square wave form or as an exponential decay (capacitative discharge) [Qin et al., (1994): (14)].
The Applicants have now developed a method for treating a colonised medium by applying a pulsed electric field to a flow of colonised medium, which method overcomes the disadvantages mentioned above (chlorination, re-colonisation, burn risk, economic constraints) even when large volumes of an aqueous medium are to be treated.
In a first aspect, the invention concerns a method for treating an aqueous flow colonised by cells by applying a pulsed electric field with an amplitude, also known as the intensity, of less than 1 kV/cm. The number of pulses applied to the cell can be of the order of 1 to 200. Preferably, the number of pulses applied is of the order of 40 to 200.
The invention also concerns the application of the method to eliminating Legionella.
In a further aspect, the invention concerns a method for destroying Legionella, characterized in that an aqueous flow colonised by Legionella is subjected to a pulsed electric field with an intensity of less than 1 kV/cm. The number of pulses applied to the cells can be of the order of 1 to 200.
Complete eradication of Legionella bacteria can be achieved by electropulsing. The bills results are obtained with low applied field intensities.